Bacterial strains and use thereof in fermentation process for 2-keto-L-gulonic acid production

ABSTRACT

The present invention relates to a process for the production of 2-keto-L-gulonic acid by fermentative conversion of L-sorbose and/or D-sorbitol. The present invention further relates to novel bacterial strains useful in this process.

FIELD OF THE INVENTION

The present invention relates to a process for the production of2-keto-L-gulonic acid by fermentative conversion of L-sorbose and/orD-sorbitol. The present invention further relates to novel bacterialstrains useful in this process.

BACKGROUND OF THE INVENTION

2-Keto-L-gulonic acid ("2-KLG") is a significant intermediate in thepreparation of L-ascorbic acid (vitamin C), an essential nutrient. 2-KLGhas been synthesized in the past on an industrial scale using theReichstein method (Helvetica Chimica Acta 17:311 (1934)). This method,however, has a number of disadvantages for commercial application,including the use of large quantities of solvents and the involvement ofa number of complex reaction steps.

Accordingly, as an alternative to the Reichstein method, a number ofprocesses employing one or more microorganisms have been developed toproduce 2-KLG by fermentation. U.S. Pat. No. 2,421,611, for example,discloses a method involving microbial oxidation of D-glucose to5-keto-D-gluconic acid, followed by chemical or microbial reduction toL-idonic acid and subsequent microbial oxidation to 2-KLG. JapanesePatent Publication Nos. 39-14493, 53-25033, 56-15877 and 59-35290, forexample, disclose similar processes involving the microbial oxidation ofD-glucose to 2,5-diketo-D-gluconic acid followed by microbial orchemical reduction to 2-KLG.

These methods, however, also suffer from a number of disadvantages thatreduce their usefulness in commercial production of 2-KLG. For example,the chemical reduction steps in these methods (i.e. the reduction of5-keto-D-gluconic acid to L-idonic acid and 2,5-diketo-D-gluconic acidto 2-KLG) are accompanied by problems with controlling thestereochemistry of reduction (thus producing D-gluconic acid and2-keto-D-gluconic acid, respectively, as byproducts) which, in turn,reduces the yield of 2-KLG. Alternatively, when this reduction isperformed by one or more microorganisms, excess sugar is required toprovide an energy source for the reduction, which also reduces the yieldof 2-KLG.

In view of these problems, an alternate pathway has been employed forthe fermentative production of 2-KLG, which involves only oxidation ofL-sorbose to 2-KLG via a sorbosone intermediate. A number of processeshave been developed using this pathway that employ a wide range ofmicroorganisms from the genera Gluconobacter, such as Gluconobacteroxydans (U.S. Pat. Nos. 4,935,359; 4,960,695; 5,312,741; and 5,541,108),Pseudogluconobacter, such as Pseudogluconobacter saccharoketogenes (U.S.Pat. No. 4,877,735; European Pat. No. 221 707), Pseudomonas, such asPseudomonas sorbosoxidans (U.S. Pat. Nos. 4,933,289 and 4,892,823), andmixtures of microorganisms from these and other genera, such asAcetobacter, Bacillus, Serratia, Mycobacterium, and Streptomyces (U.S.Pat. Nos. 3,912,592; 3,907,639; and 3,234,105).

These processes, however, suffer from certain disadvantages that limittheir usefulness for commercial production of 2-KLG. For example, theprocesses referenced above that employ G. oxydans also require thepresence of an additional "helper" microbial strain, such as Bacillusmegaterium, or commercially unattractive quantities of yeast or growthcomponents derived from yeast in order to produce sufficiently highlevels of 2-KLG for commercial use. Similarly, the processes that employPseudogluconobacter can require medium supplemented with expensive andunusual rare earth salts or the presence of a helper strain, such as B.megaterium, and/or the presence of yeast in order to achievecommercially suitable 2-KLG concentrations and efficient use of sorbosesubstrate. Other processes that employ Pseudomonas sorbosoxidans alsoinclude commercially unattractive quatities of yeast or yeast extract inthe medium.

Accordingly, there is a need in the art for microorganism strains whichefficiently produce 2-KLG, but without many of the problems associatedwith the state of the art.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to providemicroorganism strains which efficiently produce 2-KLG. Other objects,features and advantages of the present invention will be set forth inthe detailed description of preferred embodiments that follows, and inpart will be apparent from the description or may be learned by practiceof the invention. These objects and advantages of the invention will berealized and attained by the methods particularly pointed out in thewritten description and claims hereof.

These and other objects are accomplished by the methods of the presentinvention, which, in a first embodiment, is directed to a process forproducing 2-KLG from L-sorbose, which comprises the steps of culturingin a medium a microorganism of strain NRRL B-21627(ADM X6L) or a mutantor variant thereof, either alone or in mixed culture with one or morehelper strains, and then recovering the accumulated 2-KLG. Anotherembodiment of the present invention is directed to a culture of amicroorganism of strain NRRL B-21627 or a mutant thereof, such as NRRLB-21630 (ADM 86-96).

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are intended to provide further explanation of the invention asclaimed.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a depiction of the RiboPrint® patterns of bacterial strainscapable of producing 2-KLG from L-sorbose. RiboPrint® pattern (A) wasobtained from bacterial strain NRRL B-21627(ADM X6L); RiboPrint® pattern(B) was obtained from Gluconobacter oxydans strain 4025C (a reisolate ofthe small-colony component strain of the mixed culture deposit DSM 4027,U.S. Pat. No. 4,935,359); RiboPrint® pattern (C) was obtained fromPseudomonas sorbosoxidans strain IFO 14502; and RiboPrint® pattern (D)was obtained from Pseudogluconobacter saccharoketogenes strain IFO14484.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

In a first embodiment, the present invention is directed to afermentation process for the production of 2-keto-L-gulonic acid fromL-sorbose which comprises contacting a microorganism with L-sorbose fora sufficient time and then isolating the accumulated 2-KLG. Preferably,the inventive fermentation process comprises cultivating a microorganismin a synthetic or natural culture medium containing L-sorbose for asufficient time and then isolating the accumulated 2-KLG from theculture medium and/or cells of the microorganism.

The microorganism strain employed in the inventive process is preferablybacterial strain NRRL B-21627 (ADM X6L) or a mutant or variant thereof,which is capable of producing at least about 40 g/L of 2-KLG fromL-sorbose by fermentation in pure culture, i.e., in the absence of oneor more additional microorganism strain(s).

Strain NRRL B-21627 (ADM X6L) was deposited at the Agricultural ResearchService Culture Collection (NRRL), 1815 North University Street, Peoria,Ill. 61604, U.S.A., on Oct. 1, 1996 under the provisions of the BudapestTreaty and assigned accession number NRRL B-21627. The characteristicsof strain NRRL B-21627 (ADM X6L) include:

(1) Cell Morphology--gram-negative; can be gram variable in oldercultures; pleiomorphic; short rods or coccobacilli; cells appear singlyand in pairs; can form short chains or filaments; does not form spores;

(2) Colony Morphology--punctiform, convex, entire, smooth, butyrous andtranslucent; beige or light brown coloration in older colonies on somemedia;

(3) Motility: no motility observed in wet mounts prepared from liquidcultures or 2% agar plate cultures; motility observed by stabbing freshculture into a plate of BUGM™ medium (available from Biolog, Inc.,Cat.#70001) that has been partially solidified using 0.3% to 0.4% agar;cells manufacture flagella under conditions used to observe motility;

(4) Temperature range: no growth observed at 4° C., 37° C. or 41° C.,while good growth observed at 25° C., and 30° C.;

(5) pH range: no growth observed at pH 4.5; growth observed at pH 6.2;good growth observed at pH 7.2;

(6) Physiological characteristics:

(a) catalase: positive;

(b) oxidase: positive

(c) gelatinase: negative;

(d) aerobic, no growth under anaerobic conditions;

(e) brown pigment formed from fructose;

(f) acid is produced from ethanol;

(g) dihydroxyacetone is not produced from glycerol;

(h) does not form pellicle or ring within 24 hours in standing glucoseor mannitol broth culture at pH in range of 4.0-5.0; and

(i) sensitive to streptomycin; and

(7) Cultural Characteristics:

(a) growth in 3% NaCl: positive;

(b) peptone-yeast extract-mannitol agar: growth;

(c) Marine agar: slow growth;

(d) BUGM™ and BUGM-G™: growth; and

(e) Brain Heart Infusion agar: growth.

(8) RiboPrint® Analysis:

RiboPrint® analysis involves hybridization of radio-labeled anti-senseRNA to the genetic material being studied, followed by detection of thelabeled double-stranded hybrid using gel electrophoresis. The patternsobtained by this method are useful for differentiating not only betweenorganisms of different species, but also between different strains ofthe same species. RiboPrint® patterns obtained for strain NRRL B-21627(ADM X6L) and a number of comparative strains known to be capable ofproducing 2-KLG from L-sorbose are depicted in FIG. 1.

In addition to naturally occurring strain NRRL B-21627 (ADM X6L),mutants and variants thereof may also be employed in the inventiveprocess, provided that these mutants and variants are also capable ofproducing at least 40 g/L of 2-KLG from L-sorbose in monoculture.

Illustrative examples of suitable methods for preparing mutants andvariants of the inventive microorganism strains include, but are notlimited to: mutagenesis by irradiation with ultraviolet light or X-rays,or by treatment with a chemical mutagen such as nitrosoguanidine(N-methyl-N'-nitro-N-nitrosoguanidine), methylmethanesulfonate, nitrogenmustard and the like; gene integration techniques, such as thosemediated by insertional elements or transposons or by homologousrecombination of transforming linear or circular DNA molecules; andtransduction mediated by bacteriophages such as P1. These methods arewell known in the art and are described, for example, in J. H. Miller,Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1972); J. H. Miller, A Short Course inBacterial Genetics, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1992); M. Singer and P. Berg, Genes & Genomes, UniversityScience Books, Mill Valley, Calif. (1991); J. Sambrook, E. F. Fritschand T. Maniatis, Molecular Cloning: A Laboratory Manual, 2d ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); P. B.Kaufman et al., Handbook of Molecular and Cellular Methods in Biologyand Medicine, CRC Press, Boca Raton, Fla. (1995); Methods in PlantMolecular Biology and Biotechnology, B. R. Glick and J. E. Thompson,eds., CRC Press, Boca Raton, Fla. (1993); and P. F. Smith-Keary,Molecular Genetics of Escherichia coli, The Guilford Press, New York,N.Y. (1989).

Mutated strains derived from the inventive organism NRRL B-21627 (ADMX6L) using known methods are then preferably selected or screened forimproved 2-KLG production potential or for other desirable propertiesrelated to their utility in producing 2-KLG from L-sorbose. In aparticularly preferred embodiment of the mutagenesis and screeningapproach to strain improvement, mutagenized cells are selected on thebasis of their resistance to growth-inhibitory concentrations ofpartially derivatized or degraded 2-KLG , such as 2-KLG derivativesgenerated by autoclaving or other exposure to heat. In an alternativeembodiment, the selective agent may be generated by other means ofchemical modification of 2-KLG, including, but not limited to:amino-substitution to create 2-amino-L-gulonic acid or 2-amino-L-idonicacid; oxidation at the C₆ position to create 5-keto-glucaric acid;modifications leading to various thiol- or deoxy-derivatives or variousunsaturated derivatives of 2-KGL ; or by any other means that will beclear to individuals versed in the art.

A particularly preferred mutant (ADM 86-96) of strain NRRL B-21627 (ADMX6L) was deposited at the Agricultural Research Service CultureCollection (NRRL), 1815 North University Street, Peoria, Ill. 61604,U.S.A., on Oct. 15, 1996 under the provisions of the Budapest Treaty andassigned accession number NRRL B-21630.

In accordance with the present invention, the inventive microorganismstrain or a mutant or variant thereof is contacted with L-sorbose for asufficient time and then the accumulated 2-KGL is isolated. Preferably,the microorganism strain is cultivated in a natural or synthetic mediumcontaining L-sorbose for a period of time for 2-KGL to be produced andthe accumulated 2-KGL is subsequently isolated. Alternatively, apreparation derived from the cells of the microorganism strain may becontacted with L-sorbose for a sufficient time and the accumulated 2-KGLmay then be isolated.

As used herein, "a preparation derived from the cells" is intended tomean any and all extracts of cells from the culture broths of theinventive strain or a mutant or variant thereof, acetone dried cells,immobilized cells on supports, such as polyacrylamide gel, κ-carrageenanand the like, and similar preparations.

An illustrative example of such a procedure involves adding L-sorboseand CaCO₃ in a suitable aqueous buffer, such as2-(N-methylmorpholino)ethanesulfonic acid (pH 6.5; 0.5M), to an aqueousextract of the microorganism strain in a shaker flask. This reactionpreferably proceeds at a pH in the range of 5.0 to 8.0 at a temperaturein the range of 20° C. to 40° C. for about 1 to 100 hours. Theconcentration of L-sorbose should be about 0.1 to 10% w/v, morepreferably about 0.3 to 6% (w/v) and the amount of the preparationderived from the cells of strain NRRL B-21627 (ADM X6L) or a mutant orvariant thereof, such as NRRL B-21630 (ADM 86-96), should be about 1 to30 mg/ml. After shaking for a sufficient period of time undertemperature and pH conditions empirically determined to maximize 2-KGLyield, the accumulated 2-KGL may be isolated by conventional methods.

The medium used herein may be solid or liquid, synthetic (i.e. man-made)or natural, and contains sufficient nutrients for the cultivation of theinventive microorganism strain. Preferably, the medium employed is aliquid medium, more preferably a synthetic liquid medium.

In the various embodiments of the process of the present invention, thestarting material, L-sorbose, may be present in the medium prior tointroduction of the inventive microorganism strain or may be added tothe medium after introduction of the strain, either all at once at thebeginning or continuously or in installments over the course ofcultivation, or may be generated in situ by fermentative conversion ofD-sorbitol. The amount of L-sorbose employed may be determinedempirically by one skilled in the art, but is at least sufficient forthe microorganism strain to produce at least about 40 g/L of 2-KGL.Preferably, L-sorbose comprises from 3 to 30% (w/v) of the culturemedium, more preferably from 5 to 20%.

In a preferred embodiment of the present invention, the L-sorbosestarting material is generated in situ by fermentative conversion ofD-sorbitol using a suitable microorganism or mixture of microorganisms.Any microorganism or mixture of microorganisms that can convertD-sorbitol to L-sorbose in the presence of NRRL B-21627 (ADM X6L) or amutant or variant thereof while not adversely affecting its ability toconvert L-sorbose to 2-KGL may be employed. Preferably, themicroorganism employed is a strain of Gluconobacter oxydans, morepreferably G. oxydans strain ATCC 621 or G. oxydans strain IFO 3293.According to this preferred embodiment of the present invention, theD-sorbitol starting material may be present in the medium prior tointroduction of one or more of the microorganisms or may be added to themedium after introduction of one or more of the microorganisms, eitherall at once at the beginning or continuously or in installments over thecourse of cultivation.

In addition to L-sorbose and/or D-sorbitol, the natural or syntheticculture medium also contains a nitrogen source, suitable inorganicsalts, and, as appropriate, various trace nutrients, growth factors andthe like suitable for cultivation of the microorganism strain, and mayalso contain at least one supplementary carbon source. The amount ofeach of these additional ingredients to be employed is preferablyselected to maximize 2-KGL production. Such amounts may be determinedempirically by one skilled in the art according to the various methodsand techniques known in the art. In a particularly preferred embodimentof the present invention, the culture medium contains about 10% (w/v) ofL-sorbose, about 3% (wt. dry solids/v) of corn steep liquor, and about0.2% (w/v) of MgSO₄.7H₂ O, with pH controlled using NH₄ OH, Ca(OH₂) orCaCO₃. Medium for use in preparing inoculum may contain additionalcomponents as appropriate, such as peptone or N-Z Amine, supplementalcarbon sources and/or various vitamins.

Illustrative examples of suitable supplemental carbon sources include,but are not limited to: other carbohydrates, such as glucose, fructose,mannitol, starch or starch hydrolysate, cellulose hydrolysate andmolasses; organic acids, such as acetic acid, propionic acid, lacticacid, formic acid, malic acid, citric acid, and fumaric acid; andalcohols, such as glycerol.

Illustrative examples of suitable nitrogen sources include, but are notlimited to: ammonia, including ammonia gas and aqueous ammonia; ammoniumsalts of inorganic or organic acids, such as ammonium chloride, ammoniumnitrate, ammonium phosphate, ammonium sulfate and ammonium acetate;urea; nitrate or nitrite salts, and other nitrogen-containing materials,including amino acids as either pure or crude preparations, meatextract, peptone, fish meal, fish hydrolysate, corn steep liquor, caseinhydrolysate, soybean cake hydrolysate, yeast extract, dried yeast,ethanol-yeast distillate, soybean flour, cottonseed meal, and the like.

Illustrative examples of suitable inorganic salts include, but are notlimited to: salts of potassium, calcium, sodium, magnesium, manganese,iron, cobalt, zinc, copper and other trace elements, and phosphoricacid.

Illustrative examples of appropriate trace nutrients, growth factors,and the like include, but are not limited to: coenzyme A, pantothenicacid, biotin, thiamine, riboflavin, flavine mononucleotide, flavineadenine dinucleotide, other vitamins, amino acids such as cysteine,sodium thiosulfate, p-aminobenzoic acid, niacinamide, and the like,either as pure or partially purified chemical compounds or as present innatural materials. Cultivation of the inventive microorganism strain maybe accomplished using any of the submerged fermentation techniques knownto those skilled in the art, such as airlift, traditionalsparged-agitated designs, or in shaking culture.

The culture conditions employed, including temperature, pH, aerationrate, agitation rate, culture duration, and the like, may be determinedempirically by one of skill in the art to maximize 2-KGL production. Theselection of specific culture conditions depends upon factors such asthe particular inventive microorganism strain employed, mediumcomposition and type, culture technique, and similar considerations. Ina particularly preferred embodiment of the present invention whenemploying strain NRRL B-21627 (ADM X6L) or a mutant or variant thereof,such as NRRL B-21630 (ADM 86-96), cultivation takes place at atemperature in the range of 22° C. to 35° C., preferably about 30° C.,and at a pH in the range of 5.0 to 8.0, preferably in the range of 5.5to 7.5, more preferably about 6.0 to 6.8. The culture conditionsemployed can, of course, be varied by known methods at differenttimepoints during cultivation, as appropriate, to maximize 2-KGLproduction.

After cultivation for a sufficient period of time, such as, for example,from 10 to 150 hours, the 2-KGL that has accumulated in the cells and/orculture broth is isolated according to any of the known methods. Anymethod that is suitable with the conditions employed for cultivation maybe used; illustrative examples of suitable methods for recovering 2-KGLare described in U.S. Pat. Nos. 5,474,924; 5,312,741; 4,960,695;4,935,359; 4,877,735; 4,933,289; 4,892,823; 3,043,749; 3,912,592;3,907,639 and 3,234,105.

According to one such method, the microorganisms are first removed fromthe culture broth by known methods, such as centrifugation orfiltration, and the resulting solution concentrated in vacuo.Crystalline 2-KGL is then recovered by filtration and, if desired,purified by recrystallization. Similarly, 2-KGL can be recovered usingsuch known methods as the use of ion-exchange resins, solventextraction, precipitation, salting out and the like.

When 2-KGL is recovered as a free acid, it can be converted to a salt,as desired, with sodium, potassium, calcium, ammonium or similar cationsusing conventional methods. Alternatively, when 2-KGL is recovered as asalt, it can be converted to its free form or to a different salt usingconventional methods.

In an alternative embodiment of the present invention, the inventivemicroorganism is cultivated in mixed culture with one or more helperstrains. As used herein, "helper strain" is intended to mean a strain ofa microorganism that increases the amount of 2-KGL produced in theinventive process. Suitable helper strains can be determined empiricallyby one skilled in the art. Illustrative examples of suitable helperstrains include, but are not limited to, members of the followinggenera: Aureobacterium (preferably A. liquefaciens or A. saperdae),Coiynebacterium (preferably C. ammoniagenes or C. glutamicum), Bacillus,Brevibacterium (preferably B. linens or B. flavum), Pseudomonas,Proteus, Enterobacter, Citrobacter, Erwinia, Xanthomonas andFlavobacterium. Preferably, the helper strain is Corynebacteriumglutamicum ATCC 21544.

The helper strain is preferably incubated in an appropriate medium undersuitable conditions for a sufficient amount of time until a culture ofsufficient population is obtained. This helper strain inoculum may thenbe introduced into the culture medium for production of 2-KGL eitherseparately or in combination with the inventive microorganism strain,i.e., a mixed inoculum. Preferably, the ratio of the amount of thehelper strain relative to the amount of strain NRRL B-21627 (ADM X6L) isin the range of from 10:1 to 1:10,000.

Another embodiment of the present invention is directed to the novelmicroorganism strains described above which are useful in fermentationprocesses for the production of 2-KGL.

The following examples are illustrative only and are not intended tolimit the scope of the invention as defined by the appended claims. Itwill be apparent to those skilled in the art that various modificationsand variations can be made in the methods of the present inventionwithout departing from the spirit and scope of the invention. Thus, itis intended that the present invention cover the modifications andvariations of this invention provided they come within the scope of theappended claims and their equivalents.

All patents and publications referred to herein are expresslyincorporated by reference.

EXAMPLES Example 1 ISOLATION OF STRAIN NRRL B-21627 (ADM X6L)

A. ORIGIN, ENRICHMENT AND SCREENING OF SOIL SAMPLES

Environmental specimens were subjected to microbial enrichment in shakeflasks. The resulting mixed cultures were screened to identify thosecontaining at least one microorganism strain capable of producing 2-KGLfrom L-sorbose. Samples of moist soil, sand, sediment, fruit, berries,humus, and other environmental specimens were collected from variousregions of the U.S.A. Each specimen was immediately stored in a cool,ventilated, humidified container. Enrichments were started by adding onegram of soil or specimen to 30 mL of Medium A (Table 1) in a 250 mLbaffled shake flask, followed by shaking incubation at 30° C., 230 rpm,for 49 hours.

To screen the enrichments by fermentation, 0.5 to 0.75 mL of eachenrichment was transferred to a 250 mL baffled flask containing 30 mL offresh Medium B (Table 1). These flasks were shaken at 30° C., 230 rpmfor 68 hours, after which portions of the mixed culture fermentationswere analyzed for 2-KGL content, and cryogenically preserved. Forpreservation, 2.0 mL of each culture was mixed with 1.0 mL of sterile40% glycerol in water, then stored at -70° C.

Flasks were screened for 2-KGL production using thin layerchromatography on Whatman LK5 Silica Gel 150 plates, 250 mm thickness(Catalog No. 4855-820). The plates were spotted with 5 μL of centrifugedculture broth, and developed for 5-6 hours in solvent (157 mL ofn-propanol; 39 mL of deionized water; 4 mL of 1% phosphoric acid; 0.4 mLof glacial acetic acid). The plates were air dried and then sprayed with0.125 g of tetrazolium blue chloride dissolved in 25 mL of methanol and25 mL of 6N sodium hydroxide, after which they were baked at 60° C. for5 minutes. Sorbose and 2-KGL were visualized as purple spots on thefinished plates, and were identified by comparison with a standardcontaining 10 g/L each of 2-KGL and L-sorbose.

Production of 2-KGL was quantitated by HPLC. Samples were prepared by1:10 dilution in mobile phase, followed by filtration through 0.45 μmporous membranes. Mobile phase contained 1.1 mL of ACS grade sulfuricacid diluted to 4.0 L using Milli-Q water. Samples of 100 μL each wereloaded on two 2 mm×300 mm×7.8 mm Aminex HPX-87H columns (BioRad)arranged in series to provide a total column length of 600 mm, precededby a guard column of the same resin. The column was run at 55° C. usinga flow rate of 0.6 mL/min. L-sorbose and 2-KGL were detected using aWaters Model No. 410 differential refractometer, and were identified bycomparison to a standard containing 2-KGL and L-sorbose.

Thirty-three (33) of the mixed culture fermentations produced 2-KGL, inamounts ranging from 1.8 to 9.3 g/L. The mixed culture fermentation fromsoil sample #216B, from which strain NRRL B-21627 was later isolated(Example 1B), produced 6.3 g/L of 2-KGL.

B. ISOLATION AND TESTING OF MONOCULTURES

Pure cultures of microorganisms capable of producing 2-KGL fromL-sorbose, either in monoculture or in mixed culture with othermicroorganisms, were isolated from the enrichments described above.Eleven mixed-culture enrichments from Example 1A were chosen on thebasis of their superior 2-KGL production. These were thawed and dilutedin 10-fold serial increments using Medium A, after which 0.1 mL of eachdilution was spread on the surface of a Medium A agar plate. The plateswere incubated at 30° C. for 24 hours, then examined under 8- to 40-foldmagnification. Attention to the smallest, slowest-growing colonies wasnecessary in order to recover 2-KGL producing strains from the dilutionplates. Several examples of each colony type and size were selected andsubcultured on fresh Medium A plates, after which the dilution plateswere returned to 30° C. for 24 hours. Additional slow-growing colonieswere selected from the dilution plates and subcultured after the secondincubation period. Each strain was streak-purified for 1-3 cycles oneither Medium A plates or on PYM plates (10 g/L peptone; 10 g/L yeastextract; 0.5 g/L glycerol; 30 g/L mannitol; 20 g/L agar). The purestrains were cryogenically preserved at -70° C. in PYM liquid mediumcontaining 20% glycerol. A total of 118 pure strains were recovered fromthe eleven enrichment mixtures.

The 118 new strains were tested for their potential to convert L-sorboseinto 2-KGL in shake flasks. To account for the possibility that 2-KGLproduction might require the combined activity of two or moremicroorganisms, each new isolate was tested in pairwise combination withall other strains originating from the same enrichment, as well as inpure culture. To prepare inoculum, each strain was cultured on PYM agarfor 24 hours, after which a large loop of cells was suspended in sterilebuffer containing 50 mM sodium phosphate, 0.4% sodium chloride, and0.05% mannitol, pH 7.2. For each pure-strain or paired-strain test, a250 mL baffled flask containing thirty mL of Medium C (Table 1) wasinoculated with 0.2 mL of cell suspension of each of the relevantstrains. These flasks were shaken at 30° C., 230 rpm for 24 hours, afterwhich 1.0 mL was transferred to 30 mL of fermentation Medium D (Table1). The fermentation flasks were shaken at 30° C., 230 rpm for threedays, then the broth was analyzed for 2-KGL and sorbose content usingTLC and HPLC. 105 flasks were required to test the strains from soilsample #216B. Eighteen of these produced 2-KGL in amounts ranging from1.9 to 19.3 g/L, fourteen of which had been inoculated with isolate ADMX6L (Table 2). The fact that strain ADM X6L (NRRL B-21627) made 14.9 g/Lof 2-KGL in pure culture verified its identity as a 2-KGL producingstrain.

                  TABLE 1                                                         ______________________________________                                        Media employed in Example 1                                                   Ingredient (g/L)                                                                             A        B        C     D                                      ______________________________________                                        L-sorbose      20.0     50.0     20.0  50.0                                   D-glucose      2.0      0.0      1.0   0.0                                    Corn Steep Liquor dry solids                                                                 10.0     10.0     15.0  15.0                                   FeSO.sub.4.7H.sub.2 O                                                                        0.1      0.1      0.1   0.1                                    Yeast Nitrogen Base                                                                          1.7      1.7      1.7   1.7                                    CaCO.sub.3     6.0      20.0     6.0   20.0                                   Cycloheximide  0.005    0.005    0.0   0.0                                    ______________________________________                                         * The glucose, corn steep liquor, iron sulfate and calcium carbonate were     adjusted to pH 7.9, then autoclaved for 20 minutes. The remaining             ingredients were adjusted to pH 6.3, then sterilized by filtration. The       finished medium had a pH in the range of 7.1-7.4. Yeast Nitrogen Base was     Difco product #033515-9.                                                 

                  TABLE 2                                                         ______________________________________                                        2-KLG production by strain ADM X6L in pure culture and in                     mixed culture with other strains from soil sample #216-B                      216-B strain                                                                  combination                                                                             2-KLG (g/L)                                                                              L-sorbose (g/L)                                                                          % conversion yield*                           ______________________________________                                        ADM X6L   14.9       34.6       96.7                                          ADM X6L + 1                                                                             3.1        2.3        6.5                                           ADM X6L + 3                                                                             1.9        1.3        3.9                                           ADM X6L + 4                                                                             11.6       33.2       69.0                                          ADM X6L + 7                                                                             16.3       27.3       71.8                                          ADM X6L + 10                                                                            19.2       26.2       80.7                                          ADM X6L + 11a                                                                           13.6       33.1       80.5                                          ADM X6L + 11b                                                                           18.5       27.6       82.6                                          ADM X6L + 15                                                                            9.5        32.3       53.7                                          ADM X6L + 16                                                                            8.2        31.0       43.1                                          ADM X6L + 17                                                                            19.0       26.1       79.5                                          ADM X6L + 18a                                                                           19.3       24.8       76.6                                          ADM X6L + 18b                                                                           2.4        0.0        4.8                                           ADM X6L + 19                                                                            14.1       29.0       67.1                                          ______________________________________                                         *percent conversion yield refers to the weight percentage of consumed         Lsorbose that was found as 2KLG.                                         

Example 2 PRODUCTION OF 2-KLG FROM L-SORBOSE BY STRAIN NRRL B-21627 INSHAKE FLASKS

Strain NRRL B-21627 was cultured on BUGM™ solid agar medium at 30° C.,then colonies were transferred with a sterilized toothpick to a 250 mLbaffled shake flask containing 20 mL of Medium E (Table 3). The seedculture was incubated at 30° C., 240 rpm for 24 hours. Twenty mL ofproduction medium (Medium F, Medium G or Medium H; Table 3) in a 250 mLbaffled flask was then inoculated with 2 mL of this seed. The culturewas incubated at 30° C., 240 rpm for 72 hours. The broth wassubsequently withdrawn and analyzed by HPLC. The 2-KGL productionresults are shown in Table 4.

                  TABLE 3                                                         ______________________________________                                        Media employed in Example 2                                                   Ingredient (amount/liter)                                                                  E.sup.a  F.sup.b  G.sup.c                                                                              H.sup.b                                 ______________________________________                                        Yeast extract                                                                              10.0   g     15   g   0      0                                   Glycerol     5.0    mL    0.5  mL  0      5.0  mL                             MgSO.sub.4.7H.sub.2 O                                                                      0        2.5    g   2.5  g   2.5  g                              CaCO.sub.3   0        25.0   g   25.0 g   25.0 g                              L-sorbose    0        87.5   g   95.8 g   81.3 g                              Corn steep liquor dry                                                                      0        0        20.0 g   20.0 g                                solids                                                                        Bactopeptone 10.0   g     0      15.0 g   0                                   D-mannitol   0        0        14.4 g   0                                     Niacinamide (5 g/L)                                                                        0        0        9.6  mL  0                                     Thiamine     0        0        0.028                                                                              g   0                                     Pantothenic acid                                                                           0        0        0.383                                                                              g   0                                     p-aminobenzoic acid                                                                        0        0        0.192                                                                              g   0                                     D-sorbitol   20.0   g     0      0      0                                     ______________________________________                                         .sup.a pH was adjusted to 7.0 prior to autoclaving for 20 minutes             .sup.b Lsorbose was autoclaved for 20 minutes and then added to the           remaining ingredients, which had first been adjusted to pH 7.2 and            autoclaved for 20 minutes                                                     .sup.c vitamins were adjusted to pH 7.0 and sterilized by filtration, the     added to the Lsorbose and Dmannitol, which had been autoclaved for 20         minutes. This mixture was then added to the remaining ingredients, which      had first been adjusted to pH 7.2 and autoclaved for 20 minutes.         

                  TABLE 4                                                         ______________________________________                                        2-KLG Production by NRRL B-21627 in Flasks                                           MEDIUM 2-KLG (g/L)                                                     ______________________________________                                               F      18.6                                                                   G      35.6                                                                   H      27.1                                                            ______________________________________                                    

Example 3 COMPARISON OF 2-KGL PRODUCTION FROM L-SORBOSE IN PURE CULTUREBY STRAIN NRRL B-21627 RELATIVE TO OTHER STRAINS

The inventive microorganism strain NRRL B-21627 (ADM X6L) andGluconobacter oxydans strain 4025C (a reisolate of the small-colonycomponent strain of the mixed culture deposit DSM 4027, U.S. Pat. No.4,935,359) were tested for production of 2-KGL from L-sorbose usingseveral different media (Table 5) and protocols. The results (Table 6)showed significantly higher 2-KGL production by strain NRRL B-21627 in avariety of culture media. In addition, the two strains were affecteddifferently by the switch from medium I/J to medium K/L.

                  TABLE 5                                                         ______________________________________                                        Media employed in Example 3                                                   Ingredient (amount/L)                                                                      I        J        K      L                                       ______________________________________                                        N-Z Soy ™ (Sheffield)                                                                   0        0        10.0 g   0                                     ADM Arcon F ™ (ADM                                                                      0        0        0      10.0 g                                  Co.)                                                                          Corn Steep Liquor (dry                                                                     20.0   g     20.0 g   20.0 g   20.0 g                            solids)                                                                       Bactopeptone (Difco)                                                                       10.0   g     15.0 g   0      0                                   L-sorbose    0        100.0  g   0      70.0 g                                D-sorbitol   10.0   g     0      5.0  g   4.8  g                              D-mannitol   10.0   g     15.0 g   10.0 g   0                                 niacinamide  0        0.05   g   0      0.048                                                                              g                                thiamine     0        0.3    g   0      0.29 g                                pantothenic acid                                                                           0        0.4    g   0      0.386                                                                              g                                p-aminobenzoic acid                                                                        0        0.2    g   0      0.193                                                                              g                                MgSO.sub.4.7H.sub.2 O                                                                      0        2.5    g   0      2.0  g                                CaCO.sub.3   10.0   g     25.0 g   10.0 g   25.0 g                            pH (before autoclave)                                                                      6.5      7.2      6.5    7.2                                     ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Comparison of 2-KLG production by NRRL B-21627 and 4025C in                   pure culture shake flasks.                                                           Seed Medium/Fermentation Medium                                        Strain   C/D.sup.a    I/J       K/L                                           ______________________________________                                        B-21627  16.0.sup.b   39.7      33.5                                          4025C     5.4.sup.b   17.8      19.0                                          ______________________________________                                         .sup.a For media C/D (Table 1), the experimental protocol of Example 1B       was used, except that the strains were initially cultured on BUGM ™        agar, not PYM agar. For media I/J and K/L (Table 5), the experimental         protocol of Example 2 was used.                                               .sup.b 2KLG value for ADM X6L in C/D media is average of nine trials; for     4025C the value is average of six trials.                                

Example 4 PRODUCTION OF 2-KGL FROM L-SORBOSE IN SHAKE FLASKS BY MIXEDCULTURES COMPRISED OF NRRL B-21627 AND A SECOND MICROORGANISM

Baffled 250 mL shake flasks containing 20 mL of medium I were inoculatedwith 100 μL of a frozen culture of NRRL B-21627 and a like volume of asecond strain. The flasks were shaken at 240 rpm, 30° C. for 24 hours.Two mL of this culture was then transferred to a 250 mL baffled flaskcontaining 25 mL of medium M (Table 9). After 65 hours shaking at 240rpm, 30° C., the flasks were analyzed by HPLC for formation of 2-KGLfrom L-sorbose. The results are shown in Table 7.

                  TABLE 7                                                         ______________________________________                                        Production of 2-KLG from L-sorbose by mixed cultures containing               strain NRRL B-21627 (ADM X6L) in flasks.                                      Producer culture                                                                         Helper culture*                                                                            2-KLG g/L  yield %**                                  ______________________________________                                        X6L        --           28.3       100.0                                      X6L        ATCC 19354   47.7       89.9                                       X6L        ATCC 19391   42.4       82.5                                       X6L        ATCC 21544   45.6       100.0                                      X6L        ATCC 21529   41.7       96.1                                       X6L        NRRL B-14840 41.2       81.3                                       X6L        NRRL B-43647 35.6       83.4                                       ______________________________________                                         *Strains ATCC 19354, ATCC 19391, ATCC 21544, ATCC 21529, NRRL B14840, and     NRRL B43647 are Corynebacterium ammoniagenes, Brevibacterium linens,          Corynebacterium glutamicum, Brevibacterium flavum, Aureobacterium             saperdae, and Aureobacterium liquifaciens, respectively.                      **yield % refers to the weight percentage of consumed Lsorbose that was       found as 2KLG.                                                           

Example 5 MUTAGENESIS, SCREENING AND SELECTION FOR IMPROVED VARIANTS OFSTRAIN NRRL B-21627

The inventive bacterial strain NRRL B-21627 (ADM X6L) and mutantsthereof were subjected to mutagenesis, and variants showing improved2-KGL production were recovered. Bacterial cultures were grown tomid-log phase in BUGM™ or PYM broth medium, then pelleted bycentrifugation and resuspended in 2 mL of filter-steriled TM buffer in aglass tube (Tris.HCl 6.0 g/L, maleic acid 5.8 g/L, (NH₄)₂ SO₄ 1.0 g/L,Ca(NO₃)₂ 5.0 mg/L, MgSO₄.7H₂ O 0.1 g/L, FeSO₄.7H₂ O 0.25 mg/L, adjustedto pH 6.0 using KOH). The 2 mL cell suspension was mixed with 2.5 μL ofa 5.0 mg/mL solution of N'-nitro-N-nitrosoguanidine (NTG), thenincubated at 30° C. for 25 minutes. An untreated cell suspension wassimilarly incubated as a control for estimating kill rate. Afterincubation, 10 mL of TM buffer was added to each tube, then the cellswere pelleted by centrifugation, washed twice in TM buffer, thenresuspended in 4.0 mL of 0.1M NaH₂ PO₄ (phosphate buffer) adjusted to pH7.2 using KOH. The washed cell suspensions were further diluted inphosphate buffer, and aliquots were spread on plates of BUGM™ or CM2agar media then incubated at 30° C. (BUGM™ is available from Biolog,Inc., Cat.#70001; CM2 medium contained D-sorbitol 5.0 g/L, (NH₄)₂ SO₄2.0 g/L, K₂ HPO₄ 0.1 g/L, KH₂ PO₄ 0.9 g/L, FeCl₃.6H₂ O 5 mg/L, MnSO₄.4H₂O 5 mg/L, casamino acids 2.0 g/L, yeast extract 2.0 g/L, agar 15.0 g/L,pH adjusted to 7.0 prior to autoclaving; the following CM2 componentswere sterilized by filtration: MgSO₄.7H₂ O 0.25 g/L, thiamine-HCl 30mg/L, pantothenic acid and niacinamide 10 mg/L, and p-aminobenzoic acid5 mg/L). Relative to unmutagenized control cells, the kill rate from NTGtreatment was 60% to 80%. Surviving colonies were picked at random andscreened for improved 2-KGL production from L-sorbose in shake flasks.Alternatively, mutagenized cell suspensions were diluted and spread onplates of growth-inhibitory CM2-S selective agar medium (CM2-S is CM2with 7-8% 2-KGL added to the heat-sterilizable components of the mediumprior to autoclaving). Colonies that grew on CM2-S agar were picked andtested for improved potential to produce 2-KGL from L-sorbose in shakeflasks.

A. PRODUCTION OF 2-KGL FROM L-SORBOSE BY NRRL B-21627 AND MUTANTDERIVATIVES IN PURE CULTURE SHAKER FLASK FERMENTATION.

For each tested strain, 0.1 mL of frozen culture was inoculated into a250 mL baffled flask containing 20 mL seed medium K (Table 5), thenincubated at 30° C. for 24 hours at 240 rpm. 2 mL of seed contents wereused to inoculate 25 mL of fermentation medium M (Table 9) in a 250 mLbaffled shaker flask, and the flasks were shaken for 72 hours at 30° C.,240 rpm. The 2-KGL product was assayed by HPLC (Table 8).

                  TABLE 8                                                         ______________________________________                                        2-KLG production from L-sorbose by pure cultures in shaker flasks.            Strain   Type              2-KLG (g/L)                                        ______________________________________                                        X6L      wild type         27.5                                               77-111   mutant of X6L     40.6                                               86-96*   2-KLG-resistant mutant of 77-11                                                                 64.4                                               ______________________________________                                         *Strain selected on CM2S agar medium.                                    

B: PRODUCTION OF 2-KGL FROM L-SORBOSE BY NRRL B-21627 AND MUTANTDERIVATIVES IN PURE CULTURE FERMENTORS.

For each strain, a 250 mL baffled flask containing 35 mL of seed mediumN (Table 9) was inoculated with 0.2 mL of a frozen culture, thenincubated at 30° C., 240 rpm for 24 hours. 35 mL seed contents were usedto inoculate 700 mL of fermentation medium O (Table 9) in a 1.5 LApplikon fermentor. The fermentation was conducted for 72 hours underthe following controlled conditions: 32° C., 1000 rpm, pH 6.3 adjustedusing 21% NH₄ OH, aeration of 1500 mL/min. The 2-KGL product was assayedby HPLC (Table 10).

                  TABLE 9                                                         ______________________________________                                        Media employed in Examples 4 and 5                                            Ingredient (amount/L)                                                                        M         N         O                                          ______________________________________                                        N-Z Soy ™ (Sheffield)                                                                     10.0   g      5.0  g    10.0 g                                 Corn Steep Liquor (dry solids)                                                               20.0   g      10.0 g    20.0 g                                 L-sorbose      70.0   g      0       70.0 g                                   D-sorbitol     4.2    g      5.0  g    5.0  g                                 niacinamide    0.04   g      0       0.05 g                                   thiamine       0.25   g      0       0.3  g                                   pantothenic acid                                                                             0.34   g      0       0.4  g                                   p-aminobenzoic acid                                                                          0.168  g      0       0.2  g                                   MgSO.sub.4.7H.sub.2 O                                                                        2.0    g      0       2.0  g                                   CaCO.sub.3     25.0   g      5.0  g    25.0 g                                 Antifoam 6000K 0         0         0.4  mL                                    pH (before autoclave)                                                                        7.2       6.5       7.2                                        ______________________________________                                    

                  TABLE 10                                                        ______________________________________                                        2-KLG production from L-sorbose by pure cultures in fermentors.               Strain  Type         2-KLG (g/L)                                                                             molar yield %                                  ______________________________________                                        X6L     wild type    65        84                                             54-164  mutant of X6L                                                                              72.2      84                                             64-165  mutant of X6L                                                                              76.3      82                                             65-93   mutant of X6L                                                                              76        94                                             66-44   mutant of X6L                                                                              73.5      87                                             66-53   mutant of X6L                                                                              74.8      83                                             ______________________________________                                         * molar yield % refers to the molar percentage of consumed Lsorbose that      was found as 2KLG.                                                       

Example 6 2-KGL PRODUCTION FROM D-SORBITOL IN FERMENTORS BY MIXEDCULTURES COMPRISED OF ADM 86-96 (NRRL B-21630) AND A SECONDMICROORGANISM

Strain ADM 86-96 (NRRL B-21630), a mutant variant of NRRL B-21627, wascultured in the presence of a second microorganism having the capabilityof converting D-sorbitol to L-sorbose. A 500 mL baffled flask containing50 mL of seed medium P (Table 12) was inoculated with 0.2 mL of frozenculture of strain ADM 86-96 and a like volume of either ADM 29-121 (amutant of strain IFO 3293) or ATCC 621 as the second microorganism. Theflasks were shaken at 240 rpm, 30° C. for 24 hr. Seed contents were usedto inoculate 700 mL of fermentation medium Q (Table 12) in a 1.5 LApplikon fermentor. The fermentation was conducted for 43-70 hours underthe following conditions: 30° C., 800 rpm of agitation, 1.8 VVM ofaeration, pH maintained at 6.0 using 17% NH₄ OH. The results are shownin Table 11.

                  TABLE 11                                                        ______________________________________                                        2-KLG production from D-sorbitol by mixed-strain cultures in fermentors.      L-sorbose producing                                                                       2-KLG producing       % Conversion                                strain      strain      2-KLG (g/L)                                                                             Yield*                                      ______________________________________                                        ADM 29-121 (mutant                                                                        86-96       109       96                                          of IFO 3293)                                                                  ATCC 621    86-96       114       98                                          ______________________________________                                         *g 2KLG produced/(g Dsorbitol - g residual Lsorbose)                     

                  TABLE 12                                                        ______________________________________                                        Media employed in Example 6                                                   Ingredient (amount/L)                                                                            P        Q                                                 ______________________________________                                        N-Z Soy ™ (Sheffield)                                                                         10.0   g     0                                             Corn Steep Liquor (dry solids)                                                                   20.0   g     31.5   g                                      D-sorbitol         10.0   g     105.0  g                                      D-mannitol         20.0   g     0                                             niacinamide        0.05   g     0                                             thiamine           0.3    g     0                                             pantothenic acid   0.4    g     0                                             p-aminobenzoic acid                                                                              0.2    g     0                                             MgSO.sub.4.7H.sub.2 O                                                                            0        2.0      g                                        CaCO.sub.3         10.0   g     25.0   g                                      Antifoam 6000K     0        0.4      mL                                       pH (before autoclave)                                                                            6.5      7.2                                               ______________________________________                                    

What is claimed is:
 1. A process for the production of 2-keto-L-gulonicacid, which comprises culturing microorganism strain NRRL B-21627 or amutant thereof in a medium containing L-sorbose for a time sufficientfor said L-sorbose to be converted to 2-keto-L-gulonic acid; andrecovering said 2-keto-L-gulonic acid.
 2. The process according to claim1, wherein said mutant of strain NRRL B-21627 is capable of producing atleast about 40 g/L of 2-keto-L-gulonic acid from L-sorbose in pureculture.
 3. The process according to claim 1, further comprisingconverting said 2-keto-L-gulonic acid to ascorbic acid or a saltthereof.
 4. The process according to claim 1, wherein said culturing isperformed at a pH of about 5.0 to 8.0.
 5. The process according to claim1, wherein said culturing is performed at a temperature of about 22° C.to about 35° C.
 6. The process according to claim 1, wherein saidmicroorganism is cultured in pure culture.
 7. The process according toclaim 1, wherein said microorganism is cultured in a mixed culture withat least one additional microorganism strain.
 8. The process accordingto claim 7, wherein said additional microorganism strain is a member ofa genus selected from the group consisting of Aureobacterium,Corynebacterium, Bacillus, Brevibacterium, Pseudomonas, Proteus,Enterobacter, Citrobacter, Erwinia, Xanthomonas and Flavobacterium. 9.The process according to claim 8, wherein said additional microorganismstrain is Corynebacterium glutamicum.
 10. The process according to claim9, wherein said Corynebacterium glutamicum is strain ATCC
 21544. 11. Theprocess according to claim 1, wherein said L-sorbose is generated byfermentative conversion of D-sorbitol.
 12. The process according toclaim 11, wherein said L-sorbose is generated by fermentative conversionof D-sorbitol using Gluconobacter oxydans.
 13. The process according toclaim 12, wherein said Gluconobacter oxydans is strain ATCC 621 orstrain IFO 3293 or a mutant thereof.
 14. The process according to claim1, wherein said microorganism resists growth inhibition by2-keto-L-gulonic acid or chemical derivatives or degradation productsthereof.
 15. The process according to claim 1, wherein saidmicroorganism strain is NRRL B-21630.
 16. A process for the productionof 2-keto-L-gulonic acid, which comprises culturing microorganism strainNRRL B-21627 or a mutant thereof together in mixed culture with amicroorganism strain capable of converting D-sorbitol to L-sorbose in amedium containing D-sorbitol, for a time sufficient for said D-sorbitolto be converted to 2-keto-L-gulonic acid; and recovering said2-keto-L-gulonic acid.
 17. The process according to claim 16, whereinsaid additional microorganism strain is a member of the genusGluconobacter or Acetobacter.
 18. The process according to claim 17,wherein said additional microorganism strain is either Gluconobacteroxydans ATCC 621 or Gluconobacter oxydans IFO 3293 or mutants thereof.19. A biologically pure culture of microorganism strain NRRL B-21627 ora mutant thereof.
 20. The biologically pure culture according to claim19, wherein said mutant is strain NRRL B-21630.